This study is supported by the Dutch Heart Foundation and the Netherlands Institute for Regenerative Medicine.

This protocol follows the LUMC guidelines of the animal research ethics committee.
1. Preparation of Instruments, Culture Medium, and MTCS
Note: Perform all preparations in the laminar flow hood. The MTCS perfusion chamber, bubble trap and stand are described in Lieber et al., 201011.
<ol>
	<li>Disinfect the forceps and micro scissors with 70% ethanol. Prepare a 5 ml syringe with 21G needle with sterile Tyrode&#8217;s Buffer (130 mM NaCl, 5.4 m...

<ol>
	<li>Go, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+Disease+and+Stroke+Statistics--2014+Update:+A+Report+From+the+American+Heart+Association.">Heart Disease and Stroke Statistics--2014 Update: A Report From the American Heart Association.</a> Circulation. 129, e28-e292 (2013).</li><li>Gould, S. T., Srigunapalan, S., Simmons, C. A., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamic+and+Cellular+Response+Feedback+in+Calcific+Aortic+Valve+Disease.">Hemodynamic and Cellular Response Feedback in Calcific Aortic Valve Disease.</a> Circulation Research. 113 (2), 186-197 (2013).</li><li>Kruithof, B. P. T., Krawitz, S. A., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Atrioventricular+valve+development+during+late+embryonic+and+postnatal+stages+involves+condensation+and+extracellular+matrix+remodeling.">Atrioventricular valve development during late embryonic and postnatal stages involves condensation and extracellular matrix remodeling.</a> Developmental biology. 302 (1), 208-217 (2007).</li><li>Schoen, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+valves+and+valvular+pathology:+update+on+function,+disease,+repair,+and+replacement.">Cardiac valves and valvular pathology: update on function, disease, repair, and replacement.</a> Cardiovascular pathology: the official journal of the Society for Cardiovascular Pathology. 14 (4), 189-194 (2005).</li><li>Balachandran, K., Sucosky, P., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+and+mechanobiology+of+aortic+valve+inflammation+and+calcification.">Hemodynamics and mechanobiology of aortic valve inflammation and calcification.</a> International journal of inflammation. , 263870 (2011).</li><li>Butcher, J. T., Simmons, C. A., Warnock, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+of+the+Aortic+Heart+Valve.">Mechanobiology of the Aortic Heart Valve.</a> The Journal of heart valve disease. 17 (1), 62-73 (2008).</li><li>Balachandran, K., Konduri, S., Sucosky, P., Jo, H., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex+vivo+study+of+the+biological+properties+of+porcine+aortic+valves+in+response+to+circumferential+cyclic+stretch.">An ex vivo study of the biological properties of porcine aortic valves in response to circumferential cyclic stretch.</a> Annals of biomedical engineering. 34 (11), 1655-1665 (2006).</li><li>Weiler, M., Hwai Yap, C., Balachandran, K., Padala, M., Yoganathan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+analysis+of+dynamic+deformation+characteristics+of+native+aortic+valve+leaflets.">Regional analysis of dynamic deformation characteristics of native aortic valve leaflets.</a> Journal of Biomechanics. 44, 1459-1465 (2011).</li><li>Combs, M. D., Yutzey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+Regulatory+networks+in+development+and+disease.">Heart valve development: Regulatory networks in development and disease.</a> Circulation Research. 105, 408-421 (2009).</li><li>Kruithof, B. P. T., Duim, S. N., Moerkamp, A. T., Goumans, M. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF&#946;+and+BMP+signaling+in+cardiac+cushion+formation:+lessons+from+mice+and+chicken.">TGF&#946; and BMP signaling in cardiac cushion formation: lessons from mice and chicken.</a> Differentiation; research in biological diversity. 84 (1), 89-102 (2012).</li><li>Lieber, S. C., Kruithof, B. P. T., Aubry, N., Vatner, S. F., Gaussin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+a+miniature+tissue+culture+system+to+culture+mouse+heart+valves.">Design of a miniature tissue culture system to culture mouse heart valves.</a> Annals of biomedical engineering. 38 (3), 674-682 (2010).</li></ol>

Critical steps in culturing the cardiac mouse valves include making the time between excision of the heart from the mouse and the ligation in the perfusion chamber as short as possible to ensure viability and ligation of the needles perpendicular to the valves to ensure proper direction of the flow. Additionally, checking the flow after ligation in the perfusion chamber without medium ensures proper insertion and ligation of needles. It is critical to maintain a sterile culture and prevent air bubbles in the tubing, whic...

Heart valve disease is a major cause of morbidity and mortality in the Western world; its prevalence increases with age and it affects more than 10% of the population 75 years and older1. The valves of the systemic part of the heart, the aortic and mitral valves, are mostly affected. Heart valve disease is characterized by the loss of the highly organized structure of the valves, which results in the alteration of the mechanical properties2. The structural integrity is therefore critical for the function of the valve.
The leaflets of the valve are comprised of valvular interstitial cells (VIC), valvular endothelial cells (VEC), and extracellular matrix, which is highly organized in an layered pattern3,4. The VICs are responsible for the ECM synthesis, degradation and organization. Factors emanating from the bloodstream, ECs or residing in the ECM act on the VICs orchestrating its function. In addition, mechanical forces act on the leaflet during the cardiac cycle resulting in laminar or oscillatory shear stress, compressive or tensile stresses influencing the behavior of VICs5.
In order to understand how the structure of the valve is regulated, it must first be understood how VICs respond to the diverse set of stimuli experienced during the cardiac cycle. In vitro studies have been very informative about the characteristics and abilities of the valvular cells. The response of these cells in vitro, however, may not always accurately mimic the in vivo response6; for example, the response of VIC to stimuli is dependent on the presence of ECs and the ECM composition5. Furthermore, the response of the valvular cells to stimuli depends on their specific location in the leaflet7. In addition to biochemical stimuli, the behavior of the valvular cells is determined by mechanical forces acting on the valve8. Each region of the valve is subjected to its own specific set of hemodynamic stresses. Although current ex vivo models have shown that mechanical forces are important determinants of valvular structure5, the associated mechanisms are still unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development9,10, insights into adult valvular biology is still elusive.
Therefore, an ex vivo flow model was developed in which the cardiac valves can be cultured in their natural position in the heart for an extended period of time11. This has the advantage that the valves remain in their natural configuration and the VICs experience the same environment as in vivo, making the VICs responses to stimuli as natural as possible. In addition, the culture of the valves in their natural position in the heart facilitates subjecting each valvular region to the relevant hemodynamic stresses. In this ex vivo model, i.e., the Miniature Tissue Culture System (MTCS), the valves can be subjected to different biochemical and hemodynamic stimuli allowing the investigation of their role in cardiac valve remodeling.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Dulbecco&#8217;s Modified Eagle Medium</td>
			<td>life technolgies</td>
			<td>10569-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>life technolgies</td>
			<td>26140</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium</td>
			<td>life technolgies</td>
			<td>41400-045</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic</td>
			<td>life technolgies</td>
			<td>15240-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk 7-0</td>
			<td>Ethicon</td>
			<td>768G</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm culture dish</td>
			<td>Greiner bio-one</td>
			<td>664160</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tube</td>
			<td>Greiner bio-one</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>5 ml syringe</td>
			<td>BD</td>
			<td>309649</td>
			<td></td>
		</tr>
		<tr>
			<td>21 G needle</td>
			<td>BD</td>
			<td>304432</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>LEO</td>
			<td>012866-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>Tedpella</td>
			<td>1346</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon tubing</td>
			<td>Thermo Scientific</td>
			<td>8060-0020</td>
			<td>I.D. x O.D. x Wall: 1.59 x 3.18 x 0.79 mm</td>
		</tr>
		<tr>
			<td>Silicon tubing for pump</td>
			<td>Masterflex</td>
			<td>96400-13</td>
			<td>I.D. x O.D. x Wall: 0,8 x1,59 x 0,40 mm</td>
		</tr>
		<tr>
			<td>Biocompatible glue (Histoacryl)</td>
			<td>B. Braun</td>
			<td>1050071</td>
			<td></td>
		</tr>
		<tr>
			<td>precision vaporizer</td>
			<td>Dr&#228;ger</td>
			<td>Vapor 200</td>
			<td></td>
		</tr>
		<tr>
			<td>peristaltic roller pump</td>
			<td>Masterflex</td>
			<td>7521-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy-load pump head</td>
			<td>Masterflex</td>
			<td>7518-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow chamber</td>
			<td></td>
			<td></td>
			<td>see Lieber et al., 2010</td>
		</tr>
		<tr>
			<td>Bubble trap</td>
			<td></td>
			<td></td>
			<td>see Lieber et al., 2010</td>
		</tr>
	</tbody>
</table>

culturing mouse cardiac valves in the miniature tissue culture system

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the molecular, cellular and mechanical mechanisms underlying the maintenance and/or loss of the valvular structure. 
Current models used to study valvular biology include in vitro cultures of valvular endothelial and interstitial cells. Although, in vitro culturing models provide both cellular and molecular mechanisms, the mechanisms involved in the 3D-organization of the valve remain unclear. While in vivo models have provided insight into the molecular mechanisms underlying valvular development, insight into adult valvular biology is still elusive. 
In order to be able to study the regulation of the valvular 3D-organization on tissue, cellular and molecular levels, we have developed the Miniature Tissue Culture System. In this ex vivo flow model the mitral or the aortic valve is cultured in its natural position in the heart. The natural configuration and composition of the leaflet are maintained allowing the most natural response of the valvular cells to stimuli. The valves remain viable and are responsive to changing environmental conditions. This MTCS may provide advantages on studying questions including but not limited to, how does the 3D organization affect valvular biology, what factors affect 3D organization of the valve, and which network of signaling pathways regulates the 3D organization of the valve.

The aortic valve (Figure 2) or mitral valve11 can be cultured for at least 3 days. By culturing in the open position (which represents the systolic position for the aortic valve and the diastolic position for the mitral valve), valvular cells remain viable. No cell death is observed as determined by the absence of TUNEL-positive cells (Figure 2H,I) or cleaved caspase-3 expression (not shown and Lieber et al., 201011). The collagen distribution (as visualize...

Watch this Scientific Journal Video about Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System at JoVE.com

Culturing Mouse Cardiac Valves in the Miniature Tissue Culture System

Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.

Heart valve disease is a major burden in the Western world and no effective treatment is available. This is mainly due to a lack of knowledge of the ...

culturing-mouse-cardiac-valves-in-the-miniature-tissue-culture-system

Research

JoVE Journal

Bioengineering

8.7K vistas.  Leiden University Medical Center. Here, we present an ex vivo flow model in which murine cardiac valves can be cultured allowing the study of the biology of the valve.